METHOD FOR REVERSING RECENT-ONSET TYPE 1 DIABETES (T1D) BY ADMINISTERING SUBSTANCE P (sP)

ABSTRACT

Described herein is a treatment comprising the following step: (a) injecting a therapeutically effective amount of a pharmaceutical composition into the celiac artery of an individual, wherein the pharmaceutical composition reverses recent onset Type 1 Diabetes (T1D). Also described is a method for identifying an individual who will be responsive to this treatment. In addition there is described a device containing the pharmaceutical composition for injecting the pharmaceutical composition into the celiac artery.

This application claims benefit of priority to U.S. Provisional PatentApplication No. 61/886,804 filed Oct. 4, 2013, which is incorporatedherein by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to neuropeptide therapy of recent-onsetType 1 diabetes (T1D).

2. Related Art

Current methods of treatment and management of Type 1 Diabetes (T1D)include insulin replacement therapy, pancreas transplantation and isletcell transplantation. Insulin replacement therapy is a challenging anduncomfortable lifelong process plagued by side effects and it does notcure T1D. The clinical challenge is the necessity to adjust insulinrelease with control of minute-to-minute changing needs. Insulinreplacement therapy as practiced saves lives, but cannot prevent chronicsuccession of hypo- and hyperglycemic events that ultimately degrademicro- and macrovascular functionalities with broadly progressive tissuedamage and neuropathy.

SUMMARY

According to one broad aspect, the present invention provides a methodcomprising following step: (a) injecting a therapeutically effectiveamount of a pharmaceutical composition into the celiac artery of anindividual, wherein the pharmaceutical composition at least partiallyreverses recent onset Type 1 Diabetes (T1D).

According to a second broad aspect, the present invention provides adevice for injecting a pharmaceutical composition into the celiac arteryof an individual, wherein the device contains a therapeuticallyeffective amount of the pharmaceutical composition, and wherein thepharmaceutical composition at least partially reverses recent onset Type1 Diabetes (T1D).

According to a third broad aspect, the present invention provides amethod comprising the following step: (a) identifying an individual withrecent onset Type 1 Diabetes (T1D) who will respond positively toinjecting a therapeutically effective amount of a pharmaceuticalcomposition into the celiac artery of the individual, wherein thepharmaceutical composition at least partially reverses recent onset Type1 Diabetes (T1D) in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and constitutepart of this specification, illustrate exemplary embodiments of theinvention, and, together with the general description given above andthe detailed description given below, serve to explain the features ofthe invention.

FIG. 1 is a drawing showing a catheter injecting a pharmaceuticalcomposition into the celiac artery of an individual according to oneembodiment of the present invention.

FIG. 2 is a graph showing changes in blood glucose levels, in recentonset diabetic mice, following intra-arterial injection of the pancreaswith substance P (sP).

FIG. 3 is a graph illustrating the CFSE T cell proliferation assayinside the pancreatic lymph node of a transgenic, diabetic mousecarrying a diabetogenic T cell receptor (TCR).

FIG. 4 is an image of a typical, no longer infiltrated islet afterrecent onset Type 1 diabetes and intra-arterial pancreatic substance Pdelivery.

FIG. 5 is an image showing vasodilated pancreatic tissue in mice,following intra-arterial pancreas injection with substance P plus EvansBlue dye marker (observe: pancreas-selective, grayish-blue coloration).

FIG. 6 is a graph that illustrates alternative outcome models of theoverall study plan.

FIG. 7 shows a DNA collection kit that may be used in one embodiment ofthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For purposes of the present invention, a value, a determination or aproperty is “based” on a particular value, data, information, property,the satisfaction of a condition, or other factor if that value,determination or property is derived by performing a mathematicalcalculation or logical decision using that value, property or otherfactor. For example, a determination that an individual has Type 1Diabetes (T1D) based on the DNA obtained from a saliva sample from theindividual

For purposes of the present invention, the term “catheter” refers to athin tube extruded from medical grade materials that facilitates theaccess of the pancreas.

For purposes of the present invention, the term “individual” refers to amammal. For example, the term “individual” may refer to a humanindividual.

For purposes of the present invention, the term “neuropeptide” refers toa small protein-like molecule or peptide released by sensory afferentneurons to communicate with their innervated target tissue.

For purposes of the present invention, the term “recent-onset Type 1Diabetes (T1D)” refers to a clinical disease onset up to 30 months priorreversal therapy.

For purposes of the present invention, the term “respond positively toinjecting a therapeutically effective amount of a pharmaceuticalcomposition into the celiac artery of the individual” refers to anindividual for whom this injection of the pharmaceutical compositionwill at least partially reverse the effect of recent onset Type 1Diabetes (T1D) in the individual.

For purposes of the present invention, the term “therapeuticallyeffective amount” refers to an amount of substance P administered to anindividual that is effective in complete or at least partially reversingrecent-onset Type 1 Diabetes in the individual.

For purposes of the present invention, the term “Type 1 Diabetes (T1D)”refers to a form of diabetes mellitus that results from autoimmunedestruction or de-differentiation of insulin-producing beta cells of thepancreas.

DESCRIPTION

Transient receptor potential Vanilloid-1, a neuronal Ca⁺⁺-channel,TRPV1, is a major heat-sensing protein in sensory afferent or “pain”nerves. When activated, these nerves release very small biologicalmediators, neuropeptides, such as the 11-amino acid substance P. sPwidens arteries and attracts many elements of the immune system in thearea where TRPV1 was activated. It is known that TRPV1 produces amediated heat sensation. It is known that the burning sensation(generated in the brain) and the local vasodilation/hyperemia(reddening) produced by sP released by activated TRPV1 nerve endingsresults in the sensing of a heat insult (˜45° C.) to the skin: the“ouch” response learned by toddlers encountering something that is“hot”.

The sP/sP-receptor (also called NK1R−) pathway is essentially universal,with multiple, general and tissue-specific roles in diverse tissue/organcompartments. In working on the present document, it is estimated fromindependent searches, that there are more than 30,000 scientificarticles that use, measure, map sP and its cousin neuropeptide, CGRP inrelation to various in vivo or in vitro interactions and effects. Thecurrent NIH trials.gov registration site lists nearly 2000 currentclinical trials when queried with “substance P”.

sP acts as a neurotransmitter, and central neurotransmission along thesP/NK1R pathway was more recently identified as critical modifier in thepathophysiology of varied neuropsychiatric conditions, anxiety,depression, fear, phobia, migraine and diverse stress syndromes, wellbeyond its early recognized roles in acute and chronic pain, analgesia,emesis—the list of processes involving the sP/sP receptor axis in healthand disease is still growing (Haneda et al., 2007²⁹; Michelgard et al.,2007³⁰).

There is a family of TRPV-like sensors, with TRPV1 the founding and mostwidely expressed member (Dorfman et al., 2010⁴). TRPV1 integratesmultiple and diverse body bitmaps in the brain. When you tickle, break,burn or freeze your toe, the perception is sensed by TRPV1family-members, signaling to relevant brain centers, which then generatetickle or pain perception to your awareness. The toe will, however,swell because of local release of small “neuropeptides” such as sP. Theysignal tissue injury and trigger avoidance responses that favor the toefrom aggravation and repeat injury/stress.

One group of researchers that has conducted research on diabetes forseveral decades studied insulin-producing beta cells in the pancreaticislets of Langerhans almost ten years ago. These researchers werepuzzled by the observation of dense nervous system elements in- andoutside these islets (Winer et al., 2003²⁰), which float in a denselypacked mass of exocrine pancreas tissue that provides much of thedigestive enzymes. Prominent in part of this neuronal network wereabundant TRPV1+ sensory nerves whose roles were counterintuitive: whatwould TRPV1 heat sensors do in the pancreas—a pancreas temperature of45° C. is not really compatible with life.

In fact, it has been discovered (Razavi et al., 2006⁹), that in mousemodels, animals develop diabetes through progressive loss of functionalbeta cells because their TRPV1 gene has two coding mutations generatinga secretory defect, releasing only <5% of neuropeptides, including sP(Razavi et al., 2006⁹; Tsui et al., 2008¹²; Tsui et al., 2007¹⁵): sP isa growth and survival factor for beta cells, There is a local controlcircuit, where local insulin ligates insulin receptors in TRPV1terminals, which activates the channel to release local sP withoutafferent signaling to the brain. In T1D-prone mice (and also in diabetespatients, see below), beta cells for some time overcome the sP secretorydefect, inducing more sP release from “their” TRPV1+ nerve endings withinsulin. But too much insulin (called hyperinsulinism) causeshypoglycemia, low blood sugar, and the rest of the body fights that withgrowing resistance to insulin—a progressively worsening cycle thatstresses beta cells already starved for their survival factor, sP.Hyperinsulinism and insulin resistance well before disease onset aretypical for the mouse models and also for young patients with high type1 diabetes risk (Tsui et al., 2011¹⁴), in fact, it has been found thatthis is true even for adult patients with Type 2 diabetes risk (Winer etal., 2011¹⁸; Winer et al., 2009¹⁹).

Pancreatic delivery of substance P (sP) reverses recent onset Type 1diabetes mellitus (T1D) within hours in mice (Razavi et al., Cell:2006⁹). Near synchronous sP receptor binding in pancreatic islets resetsthe regulatory circuit and globally reverses beta cell stress. Thispresently proposed, initial, translational study in T1D patients, aimsto determine whether an intra-arterial (i.a.) pancreatic sP injection issafe and carries lasting promise for re-differentiation of beta cellsand -function, enhanced beta cell mass, reversing hyperinsulinism withnormalization of insulin resistance and glucose tolerance as well asdown-regulation of local diabetic autoimmunity, as it does in relevantanimal models (Tsui et al., Diabetes Metab. Res. Rev: 2011¹⁴).

Discovered about 80 years ago, substance P has been extensively used inhuman clinical studies with at best rare adverse events, in partreflecting its extremely short tissue half-life of a minute or less.Decades of experience with human clinical sP trials demonstrate its lackof toxicity. Publications of a selection of more recent clinical sPtrials, as well as analytic texts on neuronal elements of T1D, of theremission (“honeymoon”) phase of T1D and an overview of research intothe role of sP in pancreatitis are found in the Investigator's Brochure,module 1.2.3 of the CTA proposal to Health Canada, Clinical TrialsGroup.

sP is a non-toxic 11 mer neuropeptide with extremely short tissuehalf-life, released primarily by sensory afferent sensory nerves.www.trials.gov still lists nearly 2000 current trials that involve sP,sP-agonists and/or -antagonists, remarkably without sP-attributableadverse events noted there or in the older literature. Althoughreminiscent in this respect of a lack of toxicity with otherneuropeptides in clinical use (e.g. LHRH, TRH, CRH, GHRH, somatostatin),sP's short tissue half-life has so far precluded clinical utility. Inthe studies proposed here, that short half-life becomes an advantage,permitting strictly local, pancreatic drug delivery with little systemicspillage yet excellent local efficacy (Razavi et al., Cell: 2006⁹; Tsuiet al., Diabetes: 2008¹²; Tsui et al., Vanilloid Receptor TRPV1 in DrugDiscovery: Targeting Pain and Other Pathological Disorders: 2010¹³; Tsuiet al., Diabetes Metab Res Rev: 2011¹⁴; Tsui et al., Ann. N. Y. Acad.Sci.: 2008¹⁶).

These animal data demonstrate that a single sP delivery to the diabeticpancreas clears inflammatory lesions rapidly (overnight) and lastingly,through two synergistic mechanisms: 1. immediate relief of beta cellstress with lasting survival support (Razavi et al., Cell: 2006⁹) and(likely) re-activation of de-differentiated or quiescent beta cells(Talchai et al., Nat. Genet.: 2012¹⁰; Talchai et al., Cell: 2012¹¹); 2.rapid, sP-receptor-mediated, selective apoptotic elimination of recentlyactivated, inflammatory lesions in pancreas and local lymphatic tissue.I.a. pancreas injection of sP causes, like in all tissues, brief(minutes), dose-dependent hyperemia. Under experimental conditions, thiscan be expanded to a transient, pancreatitis-like reaction by precedinginjections of caerulein, a potent pancreas toxin (Koh et al., J. Cell.Mol. Med.: 2011⁷). Without such pretreatments, sP pancreas toxicity inrodents has not been observed and it has been found that even large sPdoses were not pancreas toxic in a large animal (dog) study. To extendknowledge of potential toxicity to children is a major goal of thepresent study.

Recently, this has, for essentially the first time, been achieved inanimal models, providing a strong rationale and impetus to translatethis physiological, therapeutic strategy to patients, without systemictoxicities, based on thousands of previous sP trials.

In one embodiment of the present invention, as shown in FIG. 1, acatheter 112 containing a pharmaceutical composition is used to inject atherapeutically effective amount of the pharmaceutical composition intothe celiac artery 114 of an individual to thereby partially reverserecent onset Type 1 Diabetes (T1D). Catheter 112 is attached to a supply(not shown in FIG. 1) of the pharmaceutical composition Also shown inFIG. 1 is the pancreas 122, spleen 124, stomach 126, duodenum 128,lienal artery 142, portal vein 144 and lienal vein 146.

In one embodiment of the present invention, the pharmaceuticalcomposition so injected may completely reverse recent onset T1D. In oneembodiment of the present invention, the composition may transiently formonths to years reverses recent onset T1D. In one embodiment of thepresent invention, the pharmaceutical composition aids storage,production and release of insulin by beta cells in a pancreas. In oneembodiment of the present invention, the pharmaceutical compositioncomprises a neuropeptide. In one embodiment of the present invention,the pharmaceutical composition comprises substance P. In one embodimentof the present invention, substance P is dissolved in saline in thepharmaceutical composition.

In one embodiment of the present invention, the individual treated asshown in FIG. 1 is diagnosed with Type 1 Diabetes (T1D). In oneembodiment of the present invention the individual is a human. In oneembodiment, when the individual is a human, therapeutically effectiveamount of the pharmaceutical composition is at least 10 nM/kg. In oneembodiment, when the individual is a human, therapeutically effectiveamount of the pharmaceutical composition is at least 50 nM/kg. In oneembodiment, when the individual is a human, therapeutically effectiveamount of the pharmaceutical composition is at least 100 nM/kg. In oneembodiment, when the individual is a human, therapeutically effectiveamount of the pharmaceutical composition is at least 250 nM/kg.

Unexpected neuronal elements in diabetes development have beendiscovered (Tsui et al., Rev Endocr. Metab. Disord.: 2003; Winer et al.,Nat Med: 2003¹⁷. Subsequently, it has been demonstrated (Razavi et al.,Cell: 2006) that a critical element of T1D development is pancreatic sPdeficiency, reflective of mutations in the NOD mouse TRPV1 gene. Thesemutations generate a hypofunctional/hyposecretory phenotype (Tsui etal., Vanilloid Receptor TRPV1 in Drug Discovery: Targeting Pain andOther Pathological Disorders: 2010¹³; Tsui et al., Trends Mol. Med.:2007¹⁵).

A single, pancreatic sP injection via the celiac artery reversed recentonset T1D in NOD mice for 4-6 months without any insulin therapy, aperiod approximately equivalent to about 6-8 years in humans (Tsui etal., Diabetes: 2008¹²).

Human TRPV1 is extremely polymorphic, with thousands of different allelecombinations in Caucasian and Asian populations, while it is amonomorphic gene in regions of low T1D incidence, e.g. Africa (Dorfmanet al., Vanilloid Receptor TRPV1 in Drug Discovery: Targeting Pain andOther Pathological Disorders: 2010⁴). Sequencing of over 8000 CaucasianT1D patients and controls demonstrates significant bias in TRPV1 alleleselection, which share predominantly hypofunctional phenotypes intransfection studies (unpublished interim results,Toronto-Pittsburgh-Helsinki TRPV1 consortium).

It has been directly demonstrated that systemic sP deficiency by a newlydeveloped HNK1R (sP receptor) ligation assay in the majority of T1Dpatients, finding intermediate levels in a sizable subset of firstdegree relatives (Cheung, R. K. et al., manuscript in preparation).

The fundamental link between TRPV1, beta cells and autoimmunitypreviously discovered extends to metabolic syndrome and Type 2 diabetes,emphasizing the fundamental role of neuronal controls in diabetesendocrinology (Tsui et al., Diabetes Metab Res Rev: 2011¹⁴; Winer etal., Nat Med: 2011¹⁸; Winer et al., Nat Med: 2009¹⁹).

A large animal study in dogs has recently been completed, seekingevidence for sP-induced pancreas toxicity following pancreatic injectionof sP via the celiac artery. No drug-induced toxicity was found over astudy-relevant dose range, using a broad screen of repetitivebiochemical blood tests (>1200 assays) as well as extensivehistopathology studies conducted blindly at the University of TorontoCenter for Phenogenomics by veterinary pathologists NOT associated withthe proposed trial. The injections were conducted by the Sick Kids'clinical imaging team. Since this VanilloidGenetics-sponsored study isunlikely to be published by itself, excerpt imaging data are provided inthe Protocol Addendum, showing the pancreas-selective, rapidly transient(˜7 min) sP effect as measured with a co-injected, inert blue dyemarker. The short-lived, physiological sP responses(hyperemia→pancreas→selective hyperperfusion and extravasation→normalpancreas appearance) illustrate the core effects expected to beduplicated in T1D patients.

The study has 3 stages (A, B, C), applying a cross-over design where allpatients ultimately receive the intervention: Stage A, toxicity and sPdose finding, 12 patients; Stage B, limited efficacy test, 40 randomizedpatients, cross-over design; Stage C, follow-up to 6 months, allpatients, possible extension of follow-up by 6 months.

The primary objective of Stage A is to determine if there are unexpectedadverse events with pancreatic delivery of sP in recent (<30 months)onset Type 1 Diabetic patients with basal c-Peptide levels of ≧0.2pmoles/mL. The second objective of Stage A is to determine whether sPinjection at doses of 10, 50, 100 or 250 nmoles/kg BW (3 patients perdose) improve insulin need and/or basal or stimulated c-Peptide levelscomparing pre-injection and day 20-22 post-injection mixed mealtolerance (MMTT) data. If one or more Stage A patients initiallyreceived an ineffectively small dose, an effective dose may be offeredtowards the end of the trial.

The primary objective of Stage B is to determine the prevalence ofsP-treated patients whose stimulated (MMTT) c-Peptide levels aresignificantly elevated above base line, 3 weeks post-intervention(“Responders”). The secondary objective of Stage B is to determine ifResponders show reduced insulin need, improved hyperinsulinism andimproved/normalized glycemia, 3 weeks post-intervention (MMTT data).

The primary objective of Stage C is to determine if Responders sustaintreatment effects at 6 months post intervention, or longer, if follow upis extended with a pre-planned DSMB-approved protocol change.

These study objectives acknowledge the realities of an initialtranslational effort with a stepwise approach to test its potentialclinical utility, confirm the absence of unexpected toxicities andgenerate tentative answers to two core functional questions: does humanT1D respond to sP like mouse T1D, and what is the likely longevity ofsuch effects?

The study objectives parallel previously published animal studies asclosely as possible (Razavi et al., Cell: 2006⁹; Tsui et al., VanilloidReceptor TRPV1 in Drug Discovery: Targeting Pain and Other PathologicalDisorders: 2010¹³; Tsui et al., Trends Mol. Med.: 2007¹⁵; Tsui et al.,Ann N Y Acad. Sci.: 2008¹⁶). Clinically, this post-onset study is basedon 2012 publications—an NIH-TrialNet meta-study of the first 2 yearsafter T1D onset (Greenbaum et al., Diabetes: 2012⁵) and research reportscollectively documenting the unexpectedly slow beta cell death/loss postonset (Talchai et al., Nat. Genet.: 2012¹⁰; Talchai et al., Cell:2012¹¹).

One of the several ways to confirm various conclusions about sP (Tsui,2010¹³; Tsui et al., 2008¹³) was to inject sP into the pancreas of newlydiabetic mice, a rather benign therapy as sP is a physiological,non-toxic molecule that is quite fragile and inactivated in tissue bydedicated and non-specific proteolytic degradation as well as by bindingto common NK1R in tissue within seconds. As shown in FIG. 2, sP-injecteddiabetic mice rapidly responded to the treatment, normalizing bloodsugars and other metabolic abnormalities typical for acute diabetes.Control animals injected with vehicle (saline) only rapidly succumbedfrom diabetes. In some way reminiscent of re-booting a crashed computer,sP injection had lasting effects re-establishing normal insulin controlfor months, although diabetes would eventually recur—but will againrespond to a single sP injection.

In this, the action of sP has several aspects, prominently acting stillas beta cell survival factor promoting beta cellregeneration/re-differentiation. The unexpected bonus was, however, thathemopoietic cells, including autoreactive lymphocytes that target isletsand beta cells, are for a brief time during activation highly sensitiveto sP: binding to sP receptors for a brief period sets a death signalthat kills the respective lymphocyte via a form of apoptotic, programmeddeath response. In fact, the immune system normally employs sP toterminate every-day immune responses: in inflammatory tissue undergoingan immune response, it is necessary not only to initiate but to limitthat process. FIG. 3 is a snap shot of what happens in pancreatic lymphnodes, the “breeding place” for autoimmune lymphocytes. The in vivoproliferation of these autoimmune T cells were measured as waves ofclonal expansion by cell division, each of the peaks in the figurerepresents 1 cell division inside the pancreatic lymph node: there weremany divisions in the diabetic controls (312), i.e. they rapidlygenerate many more autoimmune T cells that can kill insulin-secretingbeta cells. As shown in FIG. 3, most T cells in sP treated mice (314)did not divide at all (pink shading) in fact they die. While sP-treatedmice that also received an sP receptor (NK1R) antagonist (316) showedsurvival of at least two thirds of the pathogenic T cell clones. Thus,sP injections promote beta cell survival, regeneration andre-differentiation, while, at the same time, selectively eliminatingautoimmune cell pools that mediate beta cell death and diabetes (Razaviet al., 2006⁹; Tsui et al., 2008¹²; Tsui et al., 2007¹⁵).

In previous studies of intra-arterial (celiac artery) sP injection intorecent-onset diabetic mice (Razavi et al., 2006⁹), pancreas pathologyconsistent with pancreatitis over a dose range of 0.01-250 nM sP/kg hasnot been observed (the standard dose was 1-10 nM/kg).

FIG. 4 shows islets from mice with recent onset Type 1 diabetes. The topshows a clean, no longer inflamed islet of an sP injected mouse (1 nMsP/kg). FIG. 5 shows an infiltrated islet from a control, diabetic mouseinjected with vehicle (saline). Both islets are surrounded by normalexocrine pancreas tissue without any lesions typical for pancreatitis.

A large animal study of pancreatic sP injection in dogs, using modernpancreatitis diagnostics is currently being conducted (Mansfield et al.,2011³³) and histopathology for the detection of possible pancreatitislesions. Data are expected to be complete within about two weeks of thiswriting, in time for the anticipated Pre-CTA meeting. Dogs are arational choice of large animal experimental model, since Dogsspontaneously develop pancreatitis, have successfully been used instudies of experimental pancreatitis and well tested diagnostics areavailable (Trivedi et al., 2011³⁴).

The typical, mostly rodent, studies of pancreatitis induction that focuson the role of sP, demonstrated the vasodilation effect of the drug and,in fact, previously published sP injection data documented the same,using the commonly employed Evans Blue dye tracing strategy (Razavi etal., 2006⁹), see FIG. 5. In FIG. 5, pancreatic tissue is indicated byarrow 512.

Beyond brief, transient (minutes) tissue hyperperfusion following sPinjection, the drug does not cause tissue damage or pancreatitis-likedisease. For disease induction, the standard experimental pancreatitismodel employs multiple high dose injections of the toxic secretagoguecaerulein (Koh et al., 2011⁷).

This protocol duplicates many aspects of spontaneous pancreatitis,including elevation of both, endogenous sP and sP receptor expression inpancreatic acinar cells, the active site of pancreatitis development. Inthe exocrine pancreas, elevation of sP/sP receptor is part of the NFκBpro-inflammatory transcription pathway and begins to play a role indisease progression 3-4 days after induction with caerulein (Chan andLeung, 2011³¹; Hasel et al., 2005³⁶; Wan et al., 2008³⁵).

While sP is one of the many elements that characterize the inflammatorypancreatitis lesion, it does not elicit activation of this transcriptionpathway in acinar cells. While sP has long been a member of themulti-molecular response program caused by tissue injury, binding of sPto its receptor, NK1R, in fact can transmit a potent anti-inflammatorynet signal that explains the enhanced severity of some inflammatoryresponses to tissue damage in NK1R^(null) knock-out mice (Dib et al.,2009³²).

The injection protocol is described as: Following placing the femoralcatheter to the celiac artery under real-time imaging control, the dosefinding sP amounts or the Stage B study intervention dose (10, 50, 100or 250 nM sP/kg BW, dissolved in 5 or 10 mL saline (see above), areinjected over a 3 minute period. The rationale: Steady rate injectionwas used in all mouse studies, alternative rate injection timing werecompared in the canine study without finding any differences.

In one embodiment of the present invention, the amount of sPadministered to a patient may be 10-100 nM sP/kg BW, dissolved in 5 or10 ml saline.

In another embodiment of the present invention, the amount of sPadministered to a patient may be 100-250 nM sP/kg BW, dissolved in 5 or10 ml saline.

As shown in FIG. 6, the overall study plan is based on the assumptionthat if the intervention has effects comparable to those observed in NODmice, these should at least to some extent resemble that biology. Thus,the desired effects on T1D endocrinology should: (1) occur rapidly afterinjection of the short lived sP (612) and (2) c-Peptide (or the othermeasures (see legend of FIG. 6) should follow one or more possibleoutcomes shown (622, 624, 626, and 628).

These outcomes are measured by a mixed meal tolerance test, MMTT, on day20 post-intervention (632, 634, 636, and 638). Responders are defined tofollow lines A, B or C, non-responders, line D. All patients are then beentered into the 6 month follow up, Stage C, under standardcare—although surveillance and therapy of Responders might have to bemodified, as dictated by their T1D status.

This study is described as open-label, randomized, crossover clinicaltrial. Minimal Duration: 6 month after last patient receivedintervention; extension of Stage C: Follow-up to 12 months should beconsidered if treatment responders are identified.

Stage A is a dose finding and toxicity study. This stage determines if asingle sP dose of 10, 50, 100 or 250 nM sP/kg BW (1) generatesbiochemical signs of pancreatitis or other organ dysfunctions ineligible trial patients; or (2) improves endocrine/metabolic T1Dparameters. These doses were well tolerated in rodent and canine studies(Module 1.2.3.5). Each of these patients undergoes an MMTT 7-14d beforereceiving the intervention. The dose range was selected from animalexperience: 50-250 nmoles/kg were equally effective and lasting, 10 lessand 1 nmoles marginally so. Four patient groups of three are be injectedwith clinical trial-grade sP, in each dose group. VanilloidGenetics Inchas designed and outsourced the manufacture and validation of a clinicalgrade injectable, pediatric (sP-ped) and a regular/adolescent injectabledrug formulation (sP-R/A) in a Health Canada-approved, commercial cGMPfacility.

Stage A patients are recruited first and randomized when consented to agiven sP dose (SASS procedure)—there is not a placebo. Starting with thelowest dose, three patients receive 10 nmoles sP/kg, the next three, 50nmoles sP/kg, followed by 100 and 250 nmoles sP/kg each, in the last twogroups.

Stage B patients are randomized to the 1st or 2nd treatment group. Basedon animal data, it is expected that the maximal response to be observedat 50-100 nmoles/kg. Study physicians, together with the IGTangiographic team determines on clinical grounds if patients receive theintervention in 5 or 10 mL saline solution. At least 2 to 3 days ofobservation between groups is planned, but this period may be modifiedby study management as the clinical experience grows. Blood chemistriesrelevant to pancreas and liver and enteric function are monitored afterinjection. Patients are monitored (see below, standard care) for insulinneed and euglycemia.

The rationale behind Stage A is that Stage A generates the first patientdata. T1D reversal in recent onset diabetic mice occurs within hoursalong mechanistic pathways quite well understood. It is expected that sPeffects might be rather prompt in patients as well, given the fastkinetics of sP-receptor ligation with its positive survival effects inbeta cells and its negative survival effects on the pancreaticautoimmune-infiltrate. The Stage A strategy covers a judicious drug doserange and it proceeds cautiously—although there is no reason to expectany drug toxicities—and it generates the experience needed and the drugdose used for Stage B. The Stage A strategy and cohort size wasdiscussed and incorporated in the final minutes of a pre-CTA meetingwith Health Canada (Investigators Brochure, Module 1.2.3.1). In thispre-CTA meeting, it was also discussed that if an effective sP doseemerges in Stage A, it would be ethical to offer Stage A patients thatreceived a lower dose, a second injection at the optimal dose by the endof Stage B may be offered, about 4 weeks after study start and asscheduling allows. This does not jeopardize conclusions derived fromthis study stage or the study overall, as repeat injections aftertreatment failure are ultimately part of the follow-up plan, albeit atintervals anticipated in the range of years.

Stage B of the study is the intervention stage. Altogether 40 eligiblepatients with recent onset T1D receive the Stage A derived sP dose ofchoice by cannulation of the celiac artery under imaging guidance. Therationale: T1D reversal is an essentially binary (yes/no) initialresponse to sP therapy. Human T1D onset differs from diabetes-prone miceby the frequent (˜70%) occurrence of a transient remission phase(“honeymoon”, see below) that begins sometime in the 1st year post onsetand lasts from weeks to (sometimes) months (Module 1.2.3.3). In thepresent study, disease remission (euglycemia off insulin) might bedifficult to distinguish from spontaneous honeymoon, except that it isexpected to manifest in close proximity post-injection. While thisphase-1 study cannot be aimed to generate significant efficacy data, thecontrol cohort(s) make it possible to elucidate trends in a definitivefashion, in particular if patient treatment responses are also binaryand if sP-induced remission has anywhere near the longevity of themurine therapy (equivalent to 6-8 human years), more common relapse andre-injection after even 1 year may clinically still represent adesirable effect, in particular if non-invasive drug deliverystrategies, now in development, can be moved to application.

The next stage of the study, Stage C, is randomization. 20 eligiblepatients are recruited for randomization to intervention and 20 as thecontrol cohort in the initial intervention period, terminated in eachintervention patient 3 weeks later with an MMTT. Thus analysis in thefirst intervention group has both, pre-treatment and untreated controldata.

About 1 week after the last intervention patient was injected, thecontrol cohort crosses over to intervention status, undergo thepre-intervention MMTT, to receive the intervention about a week later:this 2nd intervention period terminates with a day 20 post-injectionMMTT. Analysis of this group relies on pre-treatment data as controls.The rationale: T1D reversal is an essentially binary (yes/no) initialresponse to sP therapy.

Approximately 70% of patients with newly diagnosed Type 1 diabetesmellitus (T1D) temporarily restore to varying extent endogenous betacell function following the initiation of insulin therapy (Bowden etal., 2008³). This period has been defined clinically in several ways,but most commonly it is now called “honeymoon” period, characterized bya daily insulin dose of <0.5 units per kg body weight per day (U/kg/d)and HbA1c≦7.0% (Bowden et al., 2008³). During honeymoon, blood glucoselevels are frequently normal and stable—usually for weeks, sometimes formonths—with little or no need for exogenous insulin and with near normalHbA1c, despite fluctuations in diet and exercise. But by 6 months pastonset, nearly all and by 12 months all but very rare patients will havereverted to pre-honeymoon insulin needs and the full typical clinicalT1D course and signs (Abdul-Rasoul et al., 2006¹).

The mechanisms governing this transiently improved beta-cell functionremain poorly understood. There is some consensus, that hyperglycemiaaround disease onset represents added beta-cell stress that functionallysilences many beta cells still surviving and that insulin therapy shouldrelieve that stress to considerable extent, sufficient for beta-cells torecover (or possibly even regenerate/re-differentiate) (van Belle etal., 2011²⁴). Considerable normalization of elevated insulin resistancearound disease onset would also contribute to improved metabolic controlin honeymoon (Schober et al., 1984²⁵), but it remains unclear how thedeclaredly auto-aggressive immune system can temporarily “shut down” itsprogressiveness: unfortunately there are few relevant data sets andthere is no animal model for honeymoon. Nevertheless, the fact thathoneymoon is common has generated consensus, that it may represent themost promising target of intervention therapies. Some 85% of new onsetcases have no T1D family history and onset is the first time diabetesdeclares itself: pre-diabetes has no obvious symptoms for many years.

Unfortunately, as aptly implied by the term, honeymoon has greatvariability in extent and duration, lasting anywhere from weeks tomonths. Researchers have observed one exceedingly rare case ofhoneymoon-like relapsing-remitting T1D that lasted well over threedecades before T1D was stable, with two of three offspring from thispatient developing full T1D early and without honeymoon (Dosch et al.unpublished). Although extremely rare, such cases illustrate thatphysiological mechanisms exist, able to curb autoimmune progression,making successful intervention strategies an ultimately attainable goal.

Because the honeymoon remission phase is a period of stable metaboliccontrol, it is important and promising to identify factors that controlthe duration of clinical remission in T1D. Several factors, includingage, gender, pubertal status, metabolic abnormalities at the time ofonset, HLA genotype, presence of diabetes-associated autoantibodies,have all been recognized to affect the likelihood of partial or completeremission in newly diabetic children (Büyükgebiz et al., 2001²¹). Fromthe discussion of published studies it becomes clear that there aredifferences in age groupings and definitions that hinder firmconclusions at this time.

The honeymoon phase often commences within days or weeks of the start ofinsulin therapy, usually lasting for weeks, rarely for months. In orderto determine frequency and duration of the honeymoon period afterinitiation of insulin therapy in newly diagnosed patients, a group of103 diabetic children, younger than 12 years of age, was prospectivelymonitored. Partial remission occurred in 69%, complete remissionoccurred in three. The length of time until remission was 28.6±12 days,the honeymoon duration was 7.2±4.8 months (Abdul-Rasoul et al., 2006¹).

A retrospective study involved 62 patients, diagnosed with T1D under theage of 18 years during the years 1991-1998 (Bowden et al., 2008³).Thirty-five patients (56.5%) entered partial remission. The length oftime until remission was 1.36±1.03 months and positively correlated withinsulin requirements at discharge from hospital.

To determine whether there are different rates of partial remission inpreschool, school-age children and adolescents with T1D, 152 consecutivepatients with newly diagnosed T1D were studied in 2004 (Bowden et al.,2008³). Patients were classified in three age-groups (group-1 (<5years), group-2 (5-12 years) and group-3 (>12 years). Clinicalcharacteristics at diagnosis, hemoglobin A1C (HbA1C) and total dailyinsulin dose (TDD) were analyzed in each age-class every three monthover 1 year. Partial remission was defined as TDD ≦0.5 units/kg/d, withHbA1C <8%. Young children (group-1, 26.8%) and adolescents (group-3,29%) had low rates of partial remission compared with school-agechildren (group-2, 56%, p=0.002). At 12 months, group differences haddisappeared: 13% ( 5/38) of group-1, 20% ( 11/56) of group-2 and 18% (8/44) of group-3 remained in partial remission.

The honeymoon period is different for each individual with T1D, thevariables that govern each course remain ill-defined and differentsmaller studies can have considerably different outcomes. Data sets wereobtained from 6,123 pediatric T1D patients (<18 years old), who weretreated in 157 pediatric centers and were observed for 36 months at thesame center starting from diagnosis (Dost et al., 2007²²) Analyses fromthis large multicenter study of diabetic children included roles forage, gender and pubertal status at onset of disease in shaping theamount of insulin required and the clinical disease remission, which inthis study occurred during the first three years of the disease.

Multiple statistical analyses were performed to identify factorsinfluencing honeymoon duration. Results revealed that partial remission(insulin <0.5 U/kg/d and HbA1c <7.0%) developed in 1992 children(32.5%), most within the first 3 months after diagnosis. Among those,21% entering remission were younger than 5 years, 37% were 5-10 yearsold, 37% of patients were 10-15 years old and 5% were adolescents, 15-17years old at disease onset. These analyses consolidate earlier reportthat the rates of partial remission are higher in younger patients.

Age at onset <5 years 5-10 years <10 years 10-15 years >15 years Boys10.4 ± 10.2 8.76 ± 9.24 9.36 ± 9.36 8.76 ± 9.24 11.04 ± 10.8  Girls 8.4± 9.2 7.56 ± 8.16  7.8 ± 8.52 9.36 ± 9.72 11.04 ± 10.08 P 0.013 0.0850.0039 0.31 0.98

Remission lasted for an average of 0.74±0.77 years (8.8 months) and wassignificantly shorter in children <10 years of age at T1D onset,compared to patients with later onset, as shown in the table above. Theshorter honeymoon phase in younger children might be related to a higherrate of ketoacidosis and/or a more abnormal metabolic situation atdiabetes onset. Children with pubertal diabetes onset had a longerremission phase, conceivably reflecting anti-inflammatory hormonaltestosterone effects, since there were gender differences in honeymoonduration, with longer remission periods in boys (estrogen is morepro-inflammatory). However, this gender effect was mainly observed inchildren <10 years old (p=0.0039), where sex hormone levels are low andno significant gender difference was found in patients with diabetesonset during or after puberty. Thus the role of hormonal effects inonset and post-onset remission remains unclear.

The extent of metabolic imbalance at disease onset seems to be anessential factor that determines the prevalence and length of remission(Abdul-Rasoul et al., 2006¹). The severity of metabolic abnormalities atthe time of diagnosis, i.e. the relatively long duration of diseaseprior treatment, large blood glucose elevations, high HbA1C values, theduration of glucosuria and high insulin requirements to establisheuglycemia (due to insulin resistance) is associated with shorterclinical remission in children with T1D (Vetter et al., 1982²³).

Diabetic ketoacidosis (DKA) at diagnosis lowers the honeymoonprevalence, perhaps reflective of a decreased capacity for beta-cellrecovery after the beginning of insulin therapy. DKA is a consequence ofinsulin deficiency, corresponding hyperglycemia with the burning offatty acids accumulating systemic ketone bodies, thus generatingpotentially fatal metabolic acidosis. Children younger than 5 years orolder than 12 years of age are more likely to develop DKA than childrenbetween 5 and 12 years of age, reasons are elusive (Bowden et al.,2008³)

Endogenous insulin secretion is assessed by measurement of C-peptide.Coded by the insulin gene, the 31 amino acid C-peptide is cleaved frompro-insulin and both are co-secreted at a one-to-one molar ratio.Measurement of baseline and stimulated C-peptide (after glucagon or amixed meal) in patients with recent-onset T1D is used as a measure ofresidual, global beta cell function or -mass. Several studies have shownthat higher C-peptide levels are positively associated with honeymoonduring the first six months of T1D and C-peptide levels are a goodpredictor of honeymoon during the first year of T1D (Zmyslowska et al.2007²⁶).

In a study of 268 patients with recently diagnosed T1D, patients werestratified by gender, age, and season at diabetes onset (Agner et al.,1987²⁷). During the first 36 months of disease, an assessment wasperformed for basal C-peptide, HbA1c, and insulin dose per kilogram.Total disease remission was set as complete discontinuation of insulintherapy for at least 1 week with stable metabolic control, while partialremission was set as an insulin need of ≦50% of the insulin dose atdischarge from the hospital. During the first 18 months of disease,12.3% of the patients developed total remission for a median of 6months, and 18.3% of patients developed partial remission, also for amedian of 6 months. Patients entering honeymoon had significantly higherbasal C-peptide levels than those who did not. From these, unfortunatelynot too consonant studies, a general conclusion can be drawn, that knownfactors predicting honeymoon and honeymoon duration include pre-pubertalonset, male gender, mild initial metabolic derangement and absence offrank ketoacidosis.

Overall, T1D honeymoon remains poorly understood, in particular withrespect to autoimmune progression and its almost certain, but unproven,transient suppression. Nevertheless, available data and broad consensusidentify the honeymoon period a natural and promising target ofintervention therapies aimed at its therapeutic extension. Isletautoimmunity does not disappear during honeymoon, as judged byT1D-associated auto-antibodies, but clearly lacks progression sincethere is sufficient endogenous beta-cell derived insulin (and c-peptide)production. The honeymoon process therefore must reflect the acuteemergence of regulatory lymphocytes which down-regulate autoimmuneeffector function—there are no data, even cues why and how that wouldoccur. The consistent failures of toxic immunosuppression trials inrecent-onset T1D patients emphasize that overall conclusion:immunosuppression kills regulatory lymphocyte pools as well as effectorcells.

Collectively, the observation of honeymoon in a large proportion ofrecent-onset T1D patients implies that physiological mechanisms forbreaking disease progression do exist and effective interventions duringthis hold promise if they support the physiological escape from diseaseprogression obvious in honeymoon. Such interventions must either slow orarrest the progression of autoimmune beta-celldestruction/de-differentiation. The substance P pancreas injection trialis the first to employ a non-toxic physiological treatment strategy inT1D which is effective in animal models to achieve just that targetprofile. It relieves the chronic neuropeptide deficiency thatcharacterizes the key genetic T1D susceptibility in TRPV1-mutant rodentsand hypofunctional TRPV1 allele selection in T1D-susceptible humans.This strategy promises to precipitate and extend honeymoon by years andrepeat injections are effective in the same animal models. The hallmarkT1D trials, DCCT/EDIC, determined that even a limited (months) period of(near) normal glucose metabolism has major impact on the devastating T1Dcomplications 2-4 decades later: while substance P therapy is not a cureof the genetic underpinnings of T1D, it promises major, positive,long-term impact on the harsh realities of living with T1D and itsenormous multi-billion annual costs long-term of complications.

In one embodiment, the amount of the sP administered to an individual isthe range of 50-100 nM/kg.

EXAMPLES Example 1 Study Cohorts—Sample Size N=52

52 eligible, consented children or adolescents aged 10-18 years arerecruited with informed, parental consent and patient assent, asappropriate. These patients have been diagnosed with T1D within ≦30months and have a basal c-Peptide level at recruitment of ≧0.2pmoles/mL.

Patients with recent onset T1D (CDA guidelineshttp://www.diabetes.ca/for-professionals/resources/2008-cpg/, seebelow), with positive measures of typical metabolic dysfunction, insulinreplacement need; T1D-associated B- & T cell autoimmunity is added, asthis T1D element is one of the direct therapy targets. The patients werediagnosed at or referred to the Hospital For Sick Children, Toronto, ON,e.g. by one of the associated SickKids Satellite Diabetes Centers in thegreater Toronto area. Patients are eligible for the trial, providedthat: 1. they are between 10-18 years old; 2. patients are ≦30 monthsfrom diagnosis and have no other chronic illnesses other than treatedhypothyroidism—not uncommon in T1D; 3. parents/caregivers and, whenagreeable to them, patients have received verbal explanations of thetrial and have viewed the Health Canada approved, educational videodescribing sP therapy in diabetes. Explanations include familiarizationwith the potential risks associated with a visceral angiogram, theinjection of sP, anesthesia and the (in humans) untested possibility ofintervention failure, prior to providing their informed consent, inwriting, for the study and the procedural angiogram.

sP treatment may not benefit patients that have normal sP secretoryactivity, controlled by the TRPV1 genotype. To stratify the cohort inthis small initial study for the most likely responders, and to reducepossible risks for unlikely responders, patients are eligible forintervention if they carry at least one of the polymorphic,T1D-associated TRPV1 alleles that are prevalent in T1Dpatients/families. Since the large TRPV1 sequencing program in Europeand North America may remain incomplete for years to come, it ispossible that sequences may be encountered that are as yet unclassified.Therefore, a bioassay has been developed that measures occupancy statusof lymphocyte NK1R (the main sP receptor which internalizes rapidlyfollowing sP-ligation). Most T1D patients have abnormally elevatedlevels of unoccupied surface NK1R, reflective of low steady state sPlevels, and this assay can be used to confirm intervention eligibility.These data are described and discussed in the HC Investigator Brochure.

Based on sequence and functional data, it is expected that as many as20% of possible recruits may fail this last, dual inclusion set which isdesigned to focus the study on the theoretically most likelysP-responsive population. Larger follow-up studies may have the power todistinguish patient subpopulations and emphasize or de-emphasize theseinclusion criteria. The rationale: T1D reversal is an essentially binary(yes/no) initial response to sP therapy. Human T1D onset differs fromdiabetes-prone mice by the frequent (˜70%) occurrence of a transientremission phase (“honeymoon”) that begins sometime in the 1st year postonset and lasts from weeks to months (Module 1.2.3.3). In the presentstudy, disease remission (euglycemia off insulin) is expected to beinduced that is difficult to distinguish from spontaneous honeymoon,except that it is expected to manifest rapidly within hours-days postinjection, rather than over a period of days. While this phase-1 studycannot be aimed to generate significant efficacy data, the controlcohort(s) makes it possible to elucidate trends in a definitive fashion,in particular if patient treatment responses are also binary and ifsP-induced remission has anywhere near the longevity of the murinetherapy (equivalent to ˜6 human years, more common relapse andre-injection after even 1 year may clinically still represent adesirable effect, in particular if the non-invasive drug deliverystrategies in development can be developed.

A number of permanent, acquired or congenital as well as transientconditions preclude participation in this study: 1. In patients with thecommon, transient remission (“honeymoon”, <0.5 U insulin/kg), there isno acute measure of drug effects and sP intervention treatment may bedelayed until disease relapses, using rising insulin needs to ≧1 U/kg asrelapse measure. 2. Patients have known co-morbidities, includingACE-inhibitor treated hypertension as well as chromosomal abnormalities,impacting one or more organ systems. Common childhood infectiousdiseases with fever ≧38° C. would lead to re-scheduling. 3. Pregnancy.4. Patients with a known radiographic contrast allergy. 5. Overweight(BMI 85-95th percentile) or obese (BMI >95th percentile for age)patients with a BMI >90th percentile for age are not be eligible forthis initial study, as c-Peptide levels are modified by elevated weight.6. Parents plan to leave Toronto imminently and follow-up cannot beassured.

Since the preclinical and animal data (Razavi et al., 2006⁹) indicatethat the treatment is most likely to benefit patients that have areduced function of TRPV1 channel. Therefore, the potential candidatemust be a carrier of at least one T1D-associated allele in the TRPV1gene: rs8065080 c.1753A>C/T/G (p.Ile585Leu/p.Ile585Phe/p.Ile585Val) or;rs224534 c.1406C>T (p.Thr469Ile) or; rs222749 c.271C>T (p.Pro91Ser) or;rs222747 c.945G>C (p.Met315Ile). Sequence listings of PCR primers foramplification and sequencing of specific TRPV1 missense variants areprovided (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8). All PCR products,500-688 bp long, are amplified from genomic DNA extracted from buccalswab samples. Purified PCR products are re-sequenced, by the certifiedGenomics Quebec facility, from both ends and the consensus genotype isdetermined. Patient genotypes are communicated only to the leadphysician who, with his advisors, confirms the eligibility for the trialbased on patient's genotype.

Prior to treatment all consented patients provide a buccal swab samplefor DNA analyses using an Oragene™ DNA Collection kit. The DNA is usedto amplify and sequence specific exons of the TRPV1 gene to identify apatient's genotypes, which will be used to determine the eligibility forthe study. Patients with wild type TRPV1 genotypes are not eligible forthis trial, as they are unlikely to benefit from the treatment and theirT1D disease might have developed due to alternative pathogenicmechanisms. FIG. 7 shows a DNA collection kit 702 that may be used inone embodiment of the present invention to collect DNA samples. DNAcollection kit 702 includes swabs 712, a tube 714, a funnel 716 thatscrews onto tube 714, a funnel lid 718 that is connected to funnel 716by a flexible strap 720, and a small cap 722 for tube 714. Although aparticular DNA collection kit is shown in FIG. 7, other types of DNAcollection kits may be used in various embodiments of the presentinvention to collect DNA samples.

Example 2 Drug Formulation

Liquid, for Injection, substance P in 0.9% Sodium Chloride, forInjection USP. Sp-R/A™: 2 mg sP/mL and sP-Ped™: 0.5 mg sP/mL, vials: 1mL each. cGMP-grade active pharmaceutical ingredient, API (PolypeptideInc, San Diego, Calif., 98% pure), cGMP sterile drug manufactureoutsourced to Dalton PharmaServices, Toronto. Production procedures andvalidation are complete and follow industry standards; manufacturingdata.

Example 3 Dosage Regimen

Dose finding and toxicity study—Stage A, 12 patients randomized toreceiving 10-50-100 or 250 nmoles/kg i.a., 3 patients per dose group.The treatment dose was originally calculated from a count of cellsurface-expressed sP receptors, NK1R, per mg mouse pancreas tissue,obtained from serial sections and extrapolated to the whole organ—not aprocedure that can be duplicated in T1D patients. The sP dose wascalculated to give a 100-fold saturation per surface receptorextrapolating to 2 nM/20 g mouse, adjusting for in-tissue dwelling timeand drug half life. This dose was indeed effective. New onset mouse T1Dhas a massive lymphocytic infiltration and many of the pancreaticsurface NK1R receptors are actually carried by these infiltrating cells,in fact, most of these cells are legitimate and almost certainlyprerequisite targets of sP therapy, as it triggers rapid lymphocytedeath in recently activated lymphocytes (Module 1.2.3.6). Thisdramatically reduces autoimmune effector cells in the pancreas and itsassociated lymph node tissue. The new onset pancreas inflammatory lesionof human T1D may be less dense, but data available are insufficient forexact comparisons, generating such data is not presently feasible. Thestandard sP dose in mouse experiments was 2 nM/mouse (i.e. 100 nm sP/kgBW) where a dose of 0.2 nM/mouse had only partial effects. On thisbackground, it is estimated that a dose of 100 nM/kg BW may provide asimilar degree of acute NK1R ligation in situ as was achieved in sPtherapy-responsive new onset T1D mice.

Example 4 Washout Period

Based on agreements during the pre-CTA meeting (May, 2012) no placebo isused, and ultimately, all patients receive an sP injection. However, ifwarranted by significant diabetes reversal rates, patients in the StageA dose finding group, who did receive an ineffectively low sP dose, maybe offered a second injection towards the end of the interventionperiod. With a vascular half-life time of as low as <30 seconds, up to aminute in some locales, the washout period for sP would be extremelyshort.)

Example 5 Pre-Study Screening and Baseline Evaluation

Standard patients care, SOP at the Hospital, includes initial as well asrepeated measurements of a number of metabolic variables, but inaddition, by 1-5 weeks prior sP intervention, at least one measurementis required of T1D-relevant B and T cell autoreactivities, as well asone MMTT. Added tests, such as the sequencing of TRPV1 allelic sequencediversity and of NK1R (sP receptor) occupancy rates was described anddiscussed in exclusion criteria, above.

Example 6 Treatment/Assessment Visits

A detailed description of the intervention and post-intervention processis provided. As described, it may be overly cautious and represent apatient stress that may well outweigh that of the intervention itself:the routine femoral injection procedure is usually done on an outpatientbase. By the end of the Stage A toxicity and dose finding period, it isdetermined if the observation period and intensive supervision can besafely reduced with DSMB agreement.

Study-relevant baseline data are important. As a rule, new onset dataare available in the patient charts. The study requires the followingblood tests, drawn 1-5 weeks before recruitment and randomization:HbA1c, T1D-associated B- and T-cell autoreactivity, pancreas and liverfunction tests and, critically, an MMTT, generating data on c-Peptide(basal, peak, AUC), fasting and stimulated insulin & glucose levels).The study aims to begin Stage A (dosing, toxicity) when 12 patients areor about to signing informed consent. Recruitment for Stage B(Intervention) continues from there.

Duplication of sampling with standard care sampling is avoided throughroutinely close coordination by the trial physician and the trialMonitor. Research Pharmacy is notified of patient scheduling, includingName, HSC ID, body weight (kg) 1-3 weeks before scheduled intervention.

The regular (adolescent) sP formulation, sP-R/A™ (BLUE label), contains2 mg sP/mL in saline, the pediatric formulation, sP-Ped™ (YELLOW label),0.5 mg sP/mL. Both formulations are stored frozen at below −20° C.

The research pharmacy receives from the study staff, patient ID, bodyweight and scheduling to re-calculate and confirm or correct theintervention dose and the appropriate number of blue or yellow vials,communicating by email with the trial physician by day minus-2. AnExcel®-based automated calculator has been developed and validated byVanilloidGenetics Inc and was provided to the study team. A writtenprescription order, stating patient Name, ID, BW and the final dose aswell as the volumes from blue-coded sP-R/A and/or yellow-coded sP-Ped tobe combined in a 10 mL sterile syringe. On the morning of intervention,the required drug vials are removed from its secure cold storage, signedout of storage, placed in a sealed, sterile plastic bag, thawed at roomtemperature and stored on water-ice until signed over to trial staff, inthis case the lead physician and the trial monitor. The ready-to-usevials are transported to the intervention OR and delivered to the IGTteam performing the injection.

On the morning of study entry (day-1), consented T1D patients have ascheduled fasting blood glucose measurement. In the rare case where arecent MMTT dataset is not available, such a patient undergoes an MMTT.After the MMTT, a modified insulin dose may be prescribed if requiredand patients are released to go home, after seeing an anesthesia and IGTconsult to discuss the next day intervention, unless these meetings havealready been concluded previously.

The next morning, fasted intervention patients are admitted to hospitalbetween 8-9 am. Provided that it has been confirmed that theintervention drug is on-hand from the Pharmacy. Patients are preparedfor and receive brief, standard anesthesia for delivery of sP via theceliac artery. The IGT imaging team has routinely carried out femoralangiograms in their clinical practice as well as performing sP celiacinjections for the entire canine study without adverse events. Stage Apatients are randomized to one of 4 groups, 3 patients each, to receive10, 50, 100 or 250 nmoles sP/kg. The given dose is injected and patientsenter a monitoring phase, initially leaving 2d between groups, aspermitted by OR availability and scheduling constraints. The datagenerated in Stage A, in particular improved fasting glucose levels andreduction in insulin need are used to identify possible sP doseresponses. All daily glucometer readings are uploaded frequently to thestudy site. The 3-4 initial (Stage A) patients are admitted overnightand then stay near the Hospital for at least the first 2 days, asdecided on clinical grounds by the PI. The conclusion of Stage-A isexpected to identify the single Stage B intervention dose, a processthat involves staff, and the study board. The rationale: Stage Agenerates the first patient data. T1D reversal in recent onset diabeticmice occurs within hours along mechanistic pathways quite wellunderstood. It is expected that sP effects might be rather prompt inpatients as well, given the fast kinetics of sP-receptor ligation withits positive survival effects in beta cells and its negative survivaleffects on the pancreatic autoimmune-infiltrate. The Stage-A strategycovers a judicious drug dose range and it proceeds cautiously althoughthere is no reason to expect any drug toxicities—and it generates theexperience needed and the drug dose used for Stage B. The stage Astrategy and cohort size was discussed, approved and incorporated in thefinal minutes of the pre-CTA meeting with Health Canada (InvestigatorsBrochure, Module 1.2.3.1). In this pre-CTA meeting, it was alsodiscussed that if an effective sP dose emerges in Stage A, it would beethical to offer Stage A patients that received a lower dose, a secondinjection at the optimal dose by the end of Stage B, about 4 weeks afterstudy start and as scheduling allows. This does not jeopardizeconclusions derived from this study stage or the study overall, asrepeat injections after treatment failure are ultimately part of thefollow-up plan, albeit at intervals anticipated in the range of years.

After completion of Stage A, these patients enter Stage C (follow up),unless it is decided to offer those patients that received a suboptimaldose a second injection with the Stage B dose. Such patients might beadded to an intervention cohort in Stage B. During completion ofinjections in Stage A, recruitment for Stage B continues: pre-setrandomization is done in groups of 5 patients each for the interventionand control cohorts. Stage B interventions begin when the first group of10 (5 controls, 5 intervention) has been consented, and it continuesuntil 40 gave consent—unless unexpected adverse events trigger a studymoratorium or DSMB-sanctioned protocol changes. The DSMB has beenconstituted with three senior, academically well respected T1Dphysicians from the U.S and one from Europe: the DSMB will communicatefrequently with the study team and as a board via Skype. Allobservations made in the Stage B toxicity and dose-finding study, aswell as any intervention-ascribed adverse events are communicatedpromptly to the DSMB, advisory board (email, phone) and adverse, inparticular sP-associated events to the REB: there now is an on-linereporting path for adverse events to Health Canada. The DSMB has thepower and obligation to stop or modify the trial in face of unexpectedadverse events. In the absence of intervention-ascribed adverse events,40 recruited and eligible patients then receive the study dose in amodified cross-over study design: 20 intervention patients arecontrolled by 20 at that point un-injected, control patients, which inturn cross over to become intervention patients, controlled by their ownpre-intervention data, 3 weeks old.

The injection protocol is described as: following catheter placementinto the celiac artery, the dose finding sP amounts or the Stage B studyintervention dose (10, 50, 100 or 250 nM sP/kg BW, dissolved in 5 or 10mL saline (see above), are injected over a 3 minute period. Therationale: Steady rate injection was used in all mouse studies,alternative rate injection timing were compared in the canine studywithout finding any differences.

After introduction of patients and parents to the study, and ifinterested in participating in the intervention and/or control group,the parents and child are seen in the Hospital IGT Clinic, in advance ofthe procedure, when a celiac angiogram is explained in detail. The risksare outlined. These include local groin issues (bleeding & hematoma,pseudoaneurysm, arterial dissection, arterial thrombosis/stenosis,AV-fistula, pain); local issues in the celiac territory (thrombosis,spasm, arterial dissection, bleeding); systemic issues (contrastallergy, X-ray exposure, air embolism, stroke); injection related(pancreatitis, liver dysfunction, cholecystitis); innovative drug status(off label). An informed consent is signed by the parents and ifappropriate, the patient assents.

Blood work is organized for the day prior to the procedure includingCBC, coagulation (INR, PTT) as well as typing and screening for 0 units,these tests are in addition to those listed above, but typing andscreening can be done several weeks prior intervention. The date of lastmenstrual period is checked for girls >12 years and pregnancy tested inaccordance with current Hospital policy. A baseline diagnostic abdominalultrasound is arranged to specifically examine the pancreas, liver, gallbladder and spleen. An anesthesia consult is organized prior to theintervention.

Example 7 Concomitant Medication

Brief anesthesia during celiac angiogram and sP injection, standardrecovery, local pain management injection site, insulin prescriptionbased on frequent blood glucose measurements.

Example 8 Efficacy Variables and Analysis

The main measurements are MMTT-derived, diabetes-associated variablesprior to and after the 3 weeks post-intervention period, the corevariable for analysis being c-Peptide (basal, peak and AUC). Secondaryanalysis includes insulin need, and estimates of insulin resistance,fasting and stimulated blood glucose—unless modified by clinicalrequirements, all values are derived from MMTTs prior to, at the end ofthe intervention and at 6- and possibly 12 months thereafter.

Example 9 Safety Variables and Analysis

Routine blood chemistries include two measurements of pancreas, liverand intestinal markers during Stage A of the study. If no abnormalvalues are observed, this number is reduced to one measurement 4-7dafter intervention.

The study follows Health Canada ICH E2A reporting Standards. Possibleadverse events are monitored and recorded by trial staff & Monitor. Twoqualified physicians, NOT part of the study team, form a stand-by AEteam, one always on home-based call, their call schedule posted to trialstaff. Minor AEs are communicated for discussion and analysis within 24hr, at most 3 hr for Serious/severe AE. All AEs are described, enteredinto study and patient records, identified as study-related/unrelated,drug treatment-related/unrelated using the SickKids adverse event form(Study Protocol, pp.: 22). Serious/severe AEs are communicated to theREB, HC and DSMB in writing.

Example 10 Statistical Analysis

This study implements recommendations of a recent NIH-TrialNet T1D metastudy, regarding clinical assay strategies, statistical approaches andinterpretations (Greenbaum et al., Diabetes: 2012⁵; Lachin et al., PLoSOne: 2011⁸). For Stage A (toxicity & dose finding), the first 12patients recruited are randomized (SAS procedure), each to one of thefour sP doses (10-50-100-250 nmoles/kg). All patients undergo apre-intervention MMTT prior intervention, the low dose first, highestlast, constituting the toxicity study. If during this dose escalationprocess, there are drug-associated, unexpected adverse events, the DSMBis consulted immediately to determine if further dose escalation shouldcontinue. Stage A will be completed with an MMTT 3 weeks after the lastStage A patient is injected, and before the start of Stage B(intervention), although recruitment, consents and pre-intervention MMTTwill be obtained earlier. Stage B (efficacy trends, n=40): In thistwo-sequence, two-period, single treatment, modified crossover study, 40patients are randomized (SAS procedure) into the initial treatment orcontrol groups (n=20 each). Patients in the treatment group undergo abaseline MMTT 7-30 days prior intervention.

These patients then receive the single-injection study intervention withthe sP dose selected in Stage A; 3 weeks later, each treated patientundergoes an MMTT. This ends Stage B for the given patient and provideall his/her data for the primary study objective. These data areclassifying that patient as responder or non-responder based onc-Peptide values and lessened insulin need, comparing the final MMTT topre-intervention data as well as relevant data sets from the controlgroup. The treated patient then crosses over to Stage C (follow up),with Home glucose measurements and insulin doses are electronicallyuploaded regularly through a secure study website and the data includedin the analysis. The frequency of glucose measurements may be determinedby the PI, as will be that of weekly-monthly phone contact between thestudy team and the family (e.g. weekly, monthly).

The (so far untreated) control group undergoes an initial MMTT duringthe second half of the initial intervention period I. After completionof this period I, control patients cross over from control to treatmentstatus and be scheduled to receive the study intervention (period II),which, for each patient, ends 3 weeks after injection of the lastpatient, and completed with an MMTT: pre- and post-intervention Data arecompared as above, the patients then crossing over to Stage C (followup). Since each patient receives only a single injection, the typicalconcerns about “wash-out” periods between the 2 periods do not apply. Inaddition, the intervention (sP) has an extremely short tissue half-life(<1 minute). Formal data analysis is performed using the GEE(generalized estimation equation) approach to build apopulation-averaged marginal model. This model accounts for correlationwithin the same patient and the time-varying covariate (treatmenteffect). The primary measure is c-Peptide, basal or stimulated, bothmeasured in MMTTs and/or major reduction or absence of insulin need.Secondary measures include insulin levels, glycemia. Differentcorrelation structures are investigated, and the most appropriatecorrelation matrices are selected.

The final model estimates the treatment, period, sequence effects, aswell as relationships between treatment response, age, age-at-onset,intervention time past onset, gender. The analysis determines if anysingle or combined marker predicts sP responsiveness/unresponsiveness.Hb1AC and autoimmune markers are compared by repeated measures ANOVApre-intervention and at 3 and 6 (and, if extended, 12) months of thestudy, as the systemic changes measured are slower to emerge thanimmediate metabolic effects. If there is consensus among the clinicalteam, advisory Board and DSMB, the follow up period may be extended,using a monitoring algorithm based on the study experience generated.The study should terminate with an MMTT for final data analysis. SAS 9.3and PROC GEMOD with repeated statement are used throughout the analysis.

The statistical power of this small initial translation study enablesanalysis in several ways. The scientific and clinical need to monitorseveral key disease markers enhances the power in combinatorialcomparisons with Bonferroni corrections where appropriate. At the6-month study endpoint, with at best very few, if any controls inhoneymoon, three sP Responders would generate a significant result. IfsP therapy in fact generates a form of honeymoon, where full remissionis defined as <0.5 U insulin/kg/d, approximately the same power wouldapply proportionately. From previous experiences in the field (Herold etal., N. Engl. J. Med.: 2002⁶), the analysis of secondary outcomes, inparticular HbA1c, c-Peptide (AUC, peak, fasting) may be most sensitiveand the several consecutive measurements in each patient addsconsiderable power and confidence in the statistical analysis despiteits small size (Greenbaum et al., Diabetes: 2012⁵; Lachin et al., PLoSOne: 2011⁸).

The brevity (3 wk) of the dosing and intervention stages, as well as thenon-intrusive character of the follow-up (Stage C, uploading ofglucometer and insulin data, 2 Hospital visits, initially bi-weeklyphone calls) is not likely to promote loss-to-follow-up, (LTF). However,LTFs might be anticipated with some preference in non-responders. Ifthis scenario begins to appear possible, despite extra efforts of thestudy team, and to enhance the statistical power of the study, standardcare Hospital chart data from up to 30 T1D patients not related to thestudy but followed by standard care at the Hospital since diagnosis overthe past 30 months are collected. Anonymized data sets on insulin use,age at onset, fasting glucose and, where available, some oral glucosetolerance test, are tabulated by a small team of students, not otherwiserelated to the study.

REFERENCES

The following references are referred to above and/or describetechnology that may be used with the present invention and contents anddisclosures of the following references are incorporated herein byreference:

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While the present invention has been disclosed with references tocertain embodiments, numerous modification, alterations, and changes tothe described embodiments are possible without departing from the sphereand scope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

What is claimed is:
 1. A method comprising the following step: (a)injecting a therapeutically effective amount of a pharmaceuticalcomposition into the celiac artery of an individual, wherein thepharmaceutical composition at least partially reverses recent onset Type1 Diabetes (T1D).
 2. The method of claim 1, wherein the therapeuticallyeffective amount of a pharmaceutical composition is injected into theceliac artery of an individual as a single dose of the pharmaceuticalcomposition.
 3. The method of claim 1, wherein the pharmaceuticalcomposition completely reverses recent onset T1D.
 4. The method of claim1, wherein the pharmaceutical composition transiently for months toyears reverses recent onset T1D.
 5. The method of claim 1, wherein thepharmaceutical composition aids storage, production and release ofinsulin by beta cells in a pancreas.
 6. The method of claim 1, whereinstep (a) is conducted for at least three minutes.
 7. The method of claim1, wherein the pharmaceutical composition comprises a neuropeptide. 8.The method of claim 1, wherein the pharmaceutical composition comprisessubstance P.
 9. The method of claim 8, wherein substance P is dissolvedin saline in the pharmaceutical composition.
 10. The method of claim 1,wherein the individual is diagnosed with Type 1 Diabetes (T1D).
 11. Themethod of claim 10, wherein the individual is diagnosed with T1D basedon DNA from a saliva sample obtained using a DNA collection kit.
 12. Themethod of claim 1, wherein the individual is a human.
 13. The method ofclaim 1, wherein the therapeutically effective amount of thepharmaceutical composition is at least 10 nM/kg.
 14. The method of claim1, wherein the therapeutically effective amount of the pharmaceuticalcomposition is at least 50 nM/kg.
 15. The method of claim 1, wherein thetherapeutically effective amount of the pharmaceutical composition is atleast 100 nM/kg.
 16. The method of claim 1, wherein the therapeuticallyeffective amount of the pharmaceutical composition is at least 250nM/kg.
 17. A device for injecting a pharmaceutical composition into theceliac artery of an individual, wherein the device contains atherapeutically effective amount of the pharmaceutical composition, andwherein the pharmaceutical composition at least partially reversesrecent onset Type 1 Diabetes (T1D).
 18. The device of claim 17, whereinthe device comprises a catheter and wherein the catheter contains thetherapeutically effective amount of the pharmaceutical composition. 19.The device of claim 17, wherein the pharmaceutical compositioncompletely reverses recent onset T1D.
 20. The device of claim 17,wherein the pharmaceutical composition transiently for months to yearsreverses recent onset T1D.
 21. The device of claim 17, wherein thepharmaceutical composition aids storage, production and release ofinsulin by beta cells in a pancreas.
 22. The device of claim 17, whereinthe pharmaceutical composition comprises a neuropeptide.
 23. The deviceof claim 17, wherein the pharmaceutical composition comprises substanceP.
 24. The device of claim 23, wherein substance P is dissolved insaline in the pharmaceutical composition.
 25. The device of claim 17,wherein the individual is diagnosed with Type 1 Diabetes (T1D).
 26. Thedevice of claim 17, wherein the individual is a human.
 27. The device ofclaim 17, wherein the therapeutically effective amount of thepharmaceutical composition is at least 10 nM/kg.
 28. The device of claim17, wherein the therapeutically effective amount of the pharmaceuticalcomposition is at least 50 nM/kg.
 29. The device of claim 17, whereinthe therapeutically effective amount of the pharmaceutical compositionis at least 100 nM/kg.
 30. The device of claim 17, wherein thetherapeutically effective amount of the pharmaceutical composition is atleast 250 nM/kg.
 31. The device of claim 17, wherein the device isinserted into the celiac artery of the individual.
 32. A methodcomprising the following step: (a) identifying an individual with recentonset Type 1 Diabetes (T1D) who will respond positively to injecting atherapeutically effective amount of a pharmaceutical composition intothe celiac artery of the individual, wherein the pharmaceuticalcomposition at least partially reverses recent onset Type 1 Diabetes(T1D) in the individual.
 33. The method of claim 32, wherein theindividual in step (a) is identified based on DNA in a saliva samplefrom the individual obtained using a DNA collection kit.